Tissue engineering combined with stem cells, an emerging field of regenerative medicine, holds promise for the restoration of tissues and organs damaged by diseases, aging, and trauma. In order to improve the therapeutic usage of stem cells, it is important to investigate ways to enhance the qualitative and quantitative extent of stem cell proliferation and differentiation, and thus increase the chances for cells to grow into desired types of tissues or structures. Temperature is an important physical factor only second to oxygen supply in its influence on growth processes in general. More specifically, in vivo study has shown that bone formation is stimulated by local diathermy. In addition, the upregulation of heat shock proteins (HSPs) and their important roles in embryonic development have been widely recognized. Local or systemic temperature elevation is common during inflammation after tissue injury and may have an important role in tissue regeneration. However, the mechanisms for temperature-induced bone cell differentiation as well as overexpression of HSPs during embryonic development are unclear. Whether or not elevated temperature affects adult stem cell recruitment and differentiation is also unknown. Although intensive investigations have been conducted on the influences of other factors, few studies have been done on thermal effects on stem cell differentiation. Therefore, the PI proposes to conduct a comprehensive study on heat shock effects on mesenchymal stem cell (MSC) proliferation and differentiation to investigate our hypothesis - that mild temperature increase may facilitate MSC differentiation and maturation. The specific aims for this two-year pilot project are: (1) to establish an optimized 3D culture condition for human mesenchymal stem cell (hMSC) proliferation using a synthetic and self-assembling peptide hydrogel - PuraMatrix, and to evaluate hMSC differentiation on 3D PuraMatrix culture versus conventional 2D culture for osteogenesis or 3D pellet culture for chondrogenesis, using classic differentiation inducers;and (2) to explore dynamic heat-shock effects on hMSC proliferation and differentiation into osteoblasts and chondrocytes in the optimized 3D culture environment, and to investigate the role of heat shock proteins (HSPs) in heat shock effects on hMSC differentiation using siRNAs targeting HSP70 and HSP27. The expected significances of this pilot study of heat shock effects on hMSC differentiation into bone and cartilage cells are: (a) to facilitate hMSC proliferation and differentiation into osteoblasts/chondrocytes;(b) to improve maturature so that differentiated cells can function as efficiently as adult cells;(c) to provide a 3D in vitro MSC culture model to maximally mimic the in vivo differentiation environment;and (d) to provide basic scientific data for the design of a thermal treatment protocol that is clinically applicable using fully differentiated stem cells in bioartifical organ/cell transplantation. PUBLIC HEALTH RELEVANCE: This pilot study may lead to a new strategy that uses thermal treatments to enhance the maturature levels of osteoblasts (bone cells) and chondrocytes (cartilage cells) differentiated from mesenchymal stem cells. With increased maturature, differentiated cells are better to perform functions similar to adult cells in tissue generation for the repair of injured or aged organs. In addition, the award of this pilot proposal will help to establish long term stem cell research in the Biomedical Engineering (BME) Department at the City College of New York (CCNY).